LU83499A1 - Blood clotting-promoting preparation based on human proteins and process for their production - Google Patents

Abstract

In a method of producing a blood-coagulation-promoting preparation based on human proteins and having a content of coagulation factors II, VII, IX and X and factor-VIII-inhibitor-bypassing-activity (FEIBA), human plasma is treated with sulphated high-polymer carbohydrates and/or with basic ion exchangers, the protein mixture with generated FEIB-activity is adsorbed on the ion exchanger, and the preparation is gained by elution and concentration.

Description

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Blood-clotting preparation based on human proteins and process for their preparation

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The invention relates to a blood coagulation-promoting preparation based on human proteins containing the coagulation factors II, VII, IX and X and a factor VIII inhibitor bypass activity.

5 '

Blood coagulation-promoting preparations with factor * VIII inhibitor bypass activity, abbreviated "FEIBA" (Factor Eight Inhibitor Bypassing Activity), are known. AT-PS 350 726 (corresponding to US Pat. No. 4,160,025 or

10 of DE-OS 27 34 821) is the production of such

Preparation described. It has been used successfully in the treatment of patients suffering from haemophilia A whose blood contains a factor VIII inhibitor (inhibitor). The chemical structure of the 15 FEIBA factor is still unknown. We only know that it is a protein with a molecular weight in the range of about 100,000. The preparation according to the abovementioned patents was produced by generation from human, citrate ion-containing plasma in the absence of free calcium ions by treatment with water-insoluble inorganic coagulation-physiological surface-active substances, such as, silica gel or kaolin, and subsequent adsorption and

Elution, whereby a mixture of the factors II, VII, IX and 25 X, the factor FEIBA and other proteins is obtained, the composition of which has not yet been described.

* - 2 - .Although, as mentioned, the preparation according to DE-OS 27 34 821 has proven to be valuable in the treatment of factor VIII inhibitor patients, the task is to expand the field of application and to improve the 5 To further improve the tolerance of FEIBA preparations, in particular to minimize undesirable side reactions such as thrombogenic and vasoactive effects.

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This object is achieved according to the invention with a blood-coagulation-promoting preparation based on human proteins containing the coagulation factors II, VII, IX and X and a factor VIII inhibitor bypass activity, which preparation is characterized by that it is free of thrombogenic activity up to 15 at least 2 units FEIBA per kg rabbit in

Thrombosis-inducing activity test according to Wessler, - that it is free from kallikrein activity and free from prekallikrein activator activity - measured in an aqueous solution of the preparation with a FEIBA-20 concentration up to at least 10 units per ml, - that it is affinity chromatographic on dextran sulfate - agarose can be separated using a NaCl gradient in such a way that the protein with factor IX activity elutes at a lower NaClr concentration than the protein 25 with FEIB activity, - that the protein with factor IX activity and the protein with Eluates containing FEIB activity in electrophoretic separation contain oo- and ß-globulins, the separation curve in the d & region comprising a main * 30 peaks corresponding to 60 to 80% of the total protein, an * adjoining shoulder of 10 to 20% of the Total protein as well as a slightly pronounced peak in the / ^ - globulin region following the shoulder-shaped course of the separation curve speaking has a content of 10 to 20% of the total protein.

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The features defined above, namely the fact that the preparation is free from thrombogenic activity and from kallikrein activity or from prekallikrein activator activity - the latter are responsible for the vasoactive effects 5 - mean that the preparation has an excellent tolerance. The thrombosis- • inducing activity test and the tests for kallikrein-

Activity and precallikrein activator activity are known to the person skilled in the art. These are specified in more detail in connection with the examples.

The third feature of the preparation according to the invention, which can be separated by affinity chromatography on dextran sulfate agarose using a NaCl gradient, is illustrated in FIG. 1 of the drawing using an example. The method of affinity chromatography on dextran sulfate agarose is known to the person skilled in the art (D.S. Peppef and C. Prowse, Thrombosis Research _1_1_ (1977), 687-692). Here, dextran sulfate (molecular weight • 20,500,000) of CNBr-activated Sepharose 4 B (Pharmacia Fine

Chemicals AB, Uppsala, Sweden). The dextran sulfate sepharose obtained in this way is equilibrated in 0.4% trisodium citrate.2H20 solution (pH 7.4) and filled into a column. The sample to be examined is applied to column 25 and then fractionally eluted with an NaCl solution in 0.4% trisodium citrate.2H2O (pH 7.4) with increasing NaCl concentration.

In Fig. 1, the fraction numbers of 30 eluates are plotted on the abscissa. The activities of the coagulation factors are plotted in units per ml on the left ordinate and the concentration of the sodium chloride gradient in mol / 1 on the right ordinate. The linear course of the gradient is drawn in as line G. As can be seen from FIG. 1, the protein with factor IX activity in the range from 0.1 to 0.5.-4- (molar), with a maximum at 0.3 (molar), elutes the protein with FEIB- Activity at a NaCl concentration in the range of 0.3 to 0.5 (molar), with a maximum at 0.4 (molar). The increasing NaCl concentration can be recognized by the NaCl-5 gradient G, which ranges from 0 molar to 0.65 molar NaCl.

Finally, for the preparation according to the invention, the fourth characteristic is the globulin content in electro-phoretic separation, which is explained in FIG. 2 of the drawing. The upper part of FIG. 2 illustrates the separation curve of the eluates according to the invention, containing the proteins with factor IX activity and FEIB activity, of dextran sulfate-Sepharose-15 chromatography using an example, whereas the lower part of FIG. 2 shows the electrophoretic separation curve of a native one Reproduces human plasma. It can be seen that in this example the main peak in the oc-globulin region is 70% of the total protein. The main summit is followed by a shoulder in the oi-globulin area, which accounts for 14% of the

Total protein content, which is followed by a weak peak after the shoulder in the f> -globulin area, which is 16% of the total protein.

Another advantage of the preparation according to the invention is that the FEIB activity is retained at least 50% after incubation in factor VIII inhibitor plasma for one hour. This property indicates a longer lasting effectiveness when applied to “30 factor VIII inhibitor patients.

*

Another property of the preparation according to the invention is advantageously that the factor IX activity is at least 50% retained for at least 35% after incubation in factor IX deficient plasma for one hour. This means that the

Preparation contains little or a very low proportion of activated factor IX. It is known that activated factor IX is inactivated in human plasma. Activated »- 5 -

Factor IX would cause adverse thrombogenic effects. From the literature (Proc. Natl. Acad. Sei. USA,

Vol. 74, No. 7, 3028-3032, July 1977, "In vitro and in vivo correlation of clotting protease activity: Effect of '5 heparin" by S.N. Gitel, R.C. Stephenson and S. Wessler) are known that factor IXa has the highest thrombogenic activity compared to other activated coagulation factors, such as Xa and Ha (thrombin).

Finally, a property of the blood-clotting preparation according to the invention is advantageously that it has an inter-alpha-trypsin inhibitor (ITI) content of 0.05 to 5 mg per FEIBA unit.

15 The ITI content causes the thrombogenicity of the preparation according to the invention to be low in the Wessler test.

The invention further comprises a method for manufacturing. Position of the new blood coagulation-promoting preparation 20 based on human proteins containing the coagulation factors II, VII, IX and X and a factor VIII inhibitor bypass activity, which method is characterized in that human plasma with sulfated high-polymer carbohydrates and / or treated with basic ion exchangers and the protein mixture is adsorbed with generated FEIB activity, after which it is obtained by elution and concentration.

When carrying out the method, care must be taken to ensure that the plasma or the reactants are kept free from substances which are capable of increasing antithrombin III activity, such as heparin or heparinoids.

According to one embodiment of the invention, the plasma 35 is first briefly treated with sulfated, high-polymer carbohydrates, the protein mixture with generated FEI.B activity is then adsorbed on an 'ion exchanger based on dextran * - 6 - \ and immediately eluted and concentrated.

According to another embodiment of the invention, the plasma is treated with a dextran-based ion exchanger and, after at least two hours of exposure, the protein mixture adsorbed on the ion exchanger is eluted with generated FEIB activity and then concentrated. In this embodiment, the generation of the FE1B-10 activity depends on the duration of exposure. This can be up to 48 hours.

The procedural measures used are not critical and can vary within wide limits; the pH-15 can be between 6 and 9, the temperature between 0 and 40 ° C, the amounts of dextran sulfate used can be 0.1 to 500 mg / 1 plasma, and DEAE-Sephadex can be 0.01 up to 10 g / 1 plasma can be used. The starting material for the method 20 according to the invention is not only native human plasma, but also plasma fractions, e.g. Cryo supernatant and the Cohn I supernatant (8% alcohol) in question.

The preparation according to the invention and the method for its production are explained in more detail in the following examples, the determination methods used being explained and the results being tabulated following the examples.

30 Example 1: 1000 1 of freshly frozen human citrate plasma are thawed at 0 to + 4 ° C and the resulting cryoprecipitate is separated off by centrifugation at + 2 ° C. The resulting. "Cryo supernatant" are added at a native pH of 7.7 to 10 g of dextran sulfate (molecular weight 500,000) and stirred for 15 minutes at + 4 ° C., the substance FEIBA being generated.

500 g of the anion exchanger DEAE-Sepha-dex A-50 (Pharmacia Fine Chemicals AB, Uppsala, Sweden) 5 are added and the mixture is stirred for half an hour at + 4 ° C., whereby the generated substance FEIBA together with the factors of the prothrombin complex ( II, VII, IX, X) and inert proteins to the insoluble DEAE Sephadex is adsorbed.

. ·. 10 The DEAE-Sephadex is separated off immediately after the adsorption process by centrifugation or filtration; the supernatant plasma can be used to obtain gamma globulin and albumin.

15 The DEAE-Sephadex is subjected to a double washing process; the DEAE-Sephadex is first treated with 50 l of a solution consisting of 4 g / 1 trisodium citrate · 21 ^ 0.7 g / 1 sodium chloride and 18 g / 1 disodium hydrogenphosphate * 12H20 in distilled water, pH 7.5 , stirred for 20 15 minutes at + 4 ° C. After separation by filtration, the DEAE-Sephadex with 50 1 of a solution consisting of 4 g / 1 trisodium citrate. · 2H'20 and 7 g / 1 sodium chloride in distilled water, pH 7.5, for 15 minutes at + 4 ° C stirred and then separated again by 25 filtration.

For elution, the DEAE-Sephadex is stirred with 25 l of a solution consisting of 30 g / 1 sodium chloride and 1 g / 1 trisodium citrate · 2H20 in distilled water, pH 7.0, for 30 minutes at + 4 ° C . The eluate containing the generated substance FEIBA, the factors of the prothrombin complex (II, VII, IX, X) and inert protein is obtained by filtration, the DEAE-Sephadex is discarded. , The eluate is dialyzed overnight against 1000 l of distilled water 35 at + 4 ° C., then frozen and subjected to a first lyophilization process. In the resulting - 8 -

Bulk material determines the FEIB activity, etc. by the method described in DE-OS 27 34 821.

For the preparation of the pharmaceutically applicable preparation with FEIB activity, the bulk material is dissolved in so much distilled, pyrogen-free water that the FEIB-> activity is between 10 and 50 FEIBA units per ml. carries (in this case 25 FEIBA units per ml). To

Adding the necessary salts to produce the isotonicity and adjusting the pH between 7.0 and 7.5, the solution is clarified by membrane filter and finally sterile filtered through a 0.2 ^ in the membrane filter. The solution is poured into the final containers at 20 ml under sterile conditions, deep-frozen and lyophilized.

15

Example 2: 1000 l of freshly frozen human citrate plasma are thawed at 0 to + 4 ° C. and the cryoprecipitate obtained is separated off by centrifugation at + 2 ° C. The resulting "cryogen supernatant" is added at a native pH of 7.7 500 g of the anion exchanger DEAE-Sepha-dex A-50 (Pharmacia Fine Chemicals AB, Uppsala, Sweden) and stirred for half an hour at + 4 ° C , the factors of the prothrombin complex (II, VII, IX, X) 25 and inert proteins being adsorbed onto the DEAE-Sephadex.

The mixture is then left to stand at + 4 ° C for 12 hours; during this "contact time" the substance • 30 FEIBA is generated.

The DEAE-Sephadex is separated after the 12 hour "contact time" by centrifugation or filtration; the supernatant plasma can be used to obtain gamma globuliri 35 and albumin.

«\ - 9 -

The further processing of the DEAE-Sephadex (two washes, elution, etc.) is carried out in the same way as described in Example 1.

5 The thrombosis-inducing activity test according to Wessler, which in the literature, etc. in J. Appl. Physiol.

14 (1959), 943-946, "Biology assay of a thrombosis-in, reducing activity in human serum" by Stanford Wessler,

Stanley M. Reimer and Mindel C. Sheps, 10 is carried out as follows: 3 rabbits are used per test. The animals are narcotized with Nembutal; after additional local anesthesia, the cardiac jugular vein is exposed, two ligatures are prepared at a distance of 1 to 2 cm.

15

The preparation to be examined is then injected in the desired dose into the ear vein opposite the exposed jugular vein within 15 seconds. The prepared ligatures are drawn within 10 to 25 seconds after the injection 20 of the preparation has ended. The isolated vein segment now remains in situ in the rabbit for 10 minutes. The piece of vein is then removed from the animal and cut open in a Petri dish in a 5% sodium citrate solution and the content is assessed according to the following scheme.

0 = no clot 1 = few macroscopically visible fibrin particles 2 = a few small thrombi 30 3 = two or more large thrombi, 4 = a single thrombus filling the entire isolated vein segment

In the case of a 4 reaction, the test is rated as positive. It is essential for the preparation according to the invention that when the preparation is injected, containing up to

V

'- 10 - to at least 2 units of FEIBA per kg of test animal, no 4-reaction occurs.

The determination of the kallikrein activity and the prekalli-5 krein activator activity is carried out in the following way.

KALLIKREIN: TO 1. Method:. Kallikrein cleaves amidolytically para-nitroaniline (pNA) from a specific chromogenic substrate. The concentration of pNA is measured photometrically at a wavelength of 405 nm.

15 2. Reagents:

Buffer: · Solution A: 3.03 g of "TRIS" and 1.7 g of imidazole are dissolved in 500 ml of 0.1N hydrochloric acid and made up to 1000 ml with water.

Solution B: 4.04 g "TRIS" and 2.27 g imidazole are dissolved in 500 ml 0.1N hydrochloric acid and made up to 1000 ml with water.

Solution C: 11.69 g of sodium chloride are dissolved in water to 25 1000 ml.

Solution A and B are mixed until a pH of 7.9 is reached. The same volume of solution C is added to this mixture.

30 Chromogenic substrate S-2302 (company Kabi, Stockholm): ‘H-D-prolyl-L-phenylalanyl-L-arginine-p-nitroanilide-dihydrochloride 1 millimolar solution of S-2302: 25 mg in 41 ml of water.

35 sample:

The sample is dissolved in the original volume and undiluted - 11 -

V

used in the test.

3rd test:

In a water bath at a temperature of 37 ° C, 5 are placed in a plastic tube ''

1.0 ml buffer, preheated to 37 ° C

0.1 ml sample “0.2 m-1 chromogenic substrate S-2302 pipetted. This mixture is introduced into a photometer at a temperature of 37 ° C. and the increase in optical density per minute (ΔΟϋ / Μΐη) at a wavelength of 405 nm, with a layer thickness of 10 mm, is measured. The activity of a sample is expressed in AOD / min.

15 PRECALLICALLY ACTIVATOR: 1st method:

Kallikrein 20 (KK) is generated from a cleaned Prekallikrein preparation (PKK) using a Prekallikrein activator (PKKA). The kallikrein cleaves amidolytically para-nitroaniline (pNA) from a specific chromogenic substrate. The concentration of pNA is measured photometrically at a wavelength of 405 nm.

25 2. Reagents: Buffer and chromogenic substrate correspond to the reagents described in the kallikrein determination.

Prekallikrein preparation: 30 The preparation is made according to a specification by Harpel, modified by M.S. Horowitz (New York Blood Center). Human citrate plasma is treated with the help of a DEAE cellulose. The fraction not bound to DEAE-Cellulo.se contains the precallikrein.

Positive control (standard): 35 i - 12 -

V

An albumin preparation from the Bureau of Biologics (BoB) of the Food and Drug Administration, Bethesda, Maryland 20205, USA is used as the standard (= reference value). This preparation contains a precallicin activator. The 5 kallikreingen generation with this BoB standard represents the reference value 1 or is set equal to 100%.

Sample:

The sample is dissolved in the original volume and used undiluted .10 in the test.

3rd test:

In a water bath at a temperature of 37 ° C. 15 0.05 ml precallikrein preparation 0.05 ml sample a) BoB standard for the reference value b) test sample (in a second test batch) are pipetted into a plastic tube. After an incubation of 10 minutes at 37 ° C., 20 0.7 ml buffer solution 0.1 ml chromogenic substrate S-2302 is pipetted. This mixture is introduced into a photometer heated to 37 ° C. and the increase in the optical density per minute (AOD / min) at a wavelength of 25 405 nm, with a layer thickness of 10 mm, is measured. The

Activity of a sample (AOD / min) is expressed factorially - compared to the BoB standard with the number 1 - or in% of the BoB standard,, 30 The characterization of the preparation according to the invention by affinity-chromatographic separation of the preparation on dextran sulfate-Sepharose has already been described in . Connection with Fig. 1 described.

35 The electrophoretic separation of the proteins with factor IX activity and with FEIB activity, which

V

2 with reference to FIG. 2 can be carried out in the following manner.

A cellulose acetate membrane, which is moistened with the electrophoresis buffer (pH 8.6, ionic strength, 0.075), serves as a support for the electrophoretic separation of the 5 different proteins. The samples to be analyzed are applied to this membrane - together with a normal human plasma as standard - and then separated in the fresh field; the separation takes place in that differently charged proteins migrate at different speeds in the electric field. For this purpose, the membrane provided with the samples is treated in a special cell filled with electrophoresis buffer for 16 to 18 minutes 15 at a voltage of 250 volts and 4 to 6 milliamperes of initial current.

After the separation process is complete, the various proteins are inserted into the fixation or

20 Staining solution made visible. After a few rinsing baths, the membrane is made transparent in another bath, applied to a glass plate and dried in a drying cabinet at 100 ° C.

25 The dried membrane is now analyzed in an automatically registering and integrating densitometer and yields separation curves, as shown in FIG. 2. The different proteins appear as bands of different strengths, the areas of which represent the relative percentage. 30 values of the corresponding proteins are proportional, whereby * these areas by the automatic integration of the

Densitometers can be determined.

The assignment of the different bands or proteins of the 35 test sample to defined proteins or protein groups is carried out by comparison with the bands of the normal human plasma analyzed as standard * - 14. The latter is broken down into 5 protein groups in the electrophoretic analysis, which by definition - in the order of decreasing migration speeds in the electric 5 field - are designated as follows: albumin, ot-globulins, / ^ - globulin, fibrinogen and jr-globulin.

The definition of the .FEIBA unit and its determination (effectiveness test) is in the literature, etc. in the 10 AT-PS 350 726 (US-PS 4,160,025, DE-OS 27 34 821) described. The determination of the activity of the coagulation factors II, VII, IX and X is also described in the aforementioned literature.

15 'The determination of the residual activity of FEIBA after incubation in factor VIII inhibitor plasma is carried out in the following manner.

1. Reagents: 20 The reagents to be used are described in connection with the determination of the FEIBA units (effectiveness test) in AT-PS 350 726 (US-PS 4,160,025, DE-OS 27 34 821).

25 2nd test:, The following dilutions are made from a preparation set to 50 FEIBA units / ml with a solution of 7 g / 1 sodium chloride and 7 g / 1 sodium citrate · 2H20 as diluent: 1: 2, 1: 4, 1: 8, ». 30 1:16, 1:32 and 1:64. Of these 6 dilutions, a 1:10 dilution is made in factor VHI inhibitor plasma (0.05 ml pre-diluted sample + 0.45 ml factor VIII Inhibitor plasma).

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- 15 -. These 1: 10 dilutions in factor VIII inhibitor plasma ("incubation mixtures") are now analyzed immediately and after an hour of incubation at 37 ° C. according to the following test scheme: 5 0.1 ml "incubation mixture" 0.1 ml phospholipid kaolin Incubate suspension at 37 ° C for 1 minute. 0.1 ml m / 40 calcium chloride

The time from the addition of calcium chloride to the formation of a clot • is measured with a stopwatch as in the effectiveness test.

3. Calculation of residual activity:

Analogous to the description in the efficacy test, a calibration curve is now created with the coagulation times of the immediately determined dilutions (undiluted sample = 50 FEIBA units / ml). The activities (FEIBA units / ml) of the various dilutions incubated for one hour are now calculated using the calibration curve and expressed as a percentage of the respective activities of the non-incubated dilutions. The mean values of the activities calculated in this way then give the average residual activity of the sample after an hour of incubation, expressed as a percentage of the starting activity before incubation.

25th

The determination of the residual activity of factor IX after incubation in factor IX deficient plasma is carried out as follows:, 30 1. Reagents:

Factor IX deficiency plasma: citrated plasma from a patient with severe hemophilia B (factor IX below 1%).

Phospholipid / kaolin suspension: PTT reagent from 35 Immuno Diagnostica Ges.m.b.H. For the test, the required amount of factor IX deficient plasma is mixed with an equal * - 16 - volume of phospholipid / kaolin suspension, incubated for 5 - minutes at 37 ° C and then kept in an ice bath during the test period.

Citrate / saline as a diluent for samples: 5 7 g / 1 trisodium citrate * 2H20, 7 g / 1 sodium chloride

Calcium chloride m / 20 (0.05 molar): Store at 37 ° C during the test period.

2nd test: 10 7 geometrical dilutions (1: 2, 1: 4 etc. to 1 128) are made from a preparation set to 50 factor IX units / ml with citrate / saline solution. A 1:10 dilution in factor IX deficient plasma is prepared from the undiluted sample and from the 7 geometric dilutions (0.05 ml pre-diluted sample + 0.45 ml factor IX deficient plasma).

These 1: 10 dilutions in factor IX deficient plasma ("incubation mixtures") are now analyzed immediately and after a 20 hour incubation at 37 ° C. according to the following test scheme, each incubation mixture being diluted with citrate / saline solution before the T: 10 determination : 0.2 ml mixture of factor IX deficient plasma and phospholi-25 pid / kaolin 0.1 ml "incubation mixture", 1:10 diluted with citrate / saline solution for 1 minute at 37 ° C '0.1 ml m / 20 calcium chloride 30

The time from calcium chloride addition to clot formation is measured with a stopwatch.

3. Calculation of the residual activity: 35 A calibration curve is created with the clotting times of the immediately determined dilutions (undiluted sample = - 17 - 50 factor IX units / ml) by doubling the clotting times against the corresponding dilutions. logarithmic graph paper. The activities (factor IX units / ml) of the various dilutions incubated for one hour are now calculated using the calibration curve and as a percentage of the respective activity. of the non-incubated dilutions. The mean values of the activities calculated in this way then give the average residual activity of the sample after a 10 hour incubation, expressed as a percentage of the starting activity before incubation.

Immunological determination of the inter-alpha-trypsin inhibitor (ITI): 15 1st method:

The determination is carried out using the Ouchterlony technique, whereby a specific antibody against an antigen-containing sample diffuses in an agar medium. The antigen reacts specifically with the antibody and forms an immunoprecipitation band, which is considered a positive reaction.

2. Reagents:

Antiserum against rabbit ITI, Behringwerke AG, Mar-25 burg / Lahn, FRG

Agar: A solution of 1.25 g agar, 0.9 g sodium chloride and 100 mg sodium azide in 100 ml water is briefly boiled and the hot, homogeneous solution is poured into plates in a layer thickness of approx. 2 mm. In the cooled, ver. 30 solidified gels are punched in 2 rows at a distance of 5 mm and approximately 2 mm large holes.

Standard or sample:

A protein standard serum from Behringwerke with a defined ITI content serves as the calibration substance. A geometric series of dilutions in physiological sodium chloride solution (9 g.-18- ».

NaCl / 1).

The sample to be tested is operated as with the standard.

5 3rd test:

The dilutions of the calibration substance or the sample to be tested are introduced into a row of holes in the agar, and the undiluted specific antiserum is pipetted into the row of holes next to it. The agar-10 plate charged in this way is incubated at 37 ° C. for 15 hours. The immunoprecipitations are then read.

4. Calculation of the ITI concentration:

The yardstick for the ITI concentration of a sample is the dilution level at which precipitation is just still visible ("titer" of the sample). The ITI concentration of the sample to be tested is calculated as follows: l-iter.der., Test sample iTi concentration of the standard 20 titer of the standard

The ITI concentration is given in mg% (mg / 100 ml).

The preparations prepared according to Examples 1 and 2 had the following characteristic values after carrying out the above determination methods:

The representations in FIGS. 1 and 2 of the drawing correspond with respect to the affinity chromatographic and electrophoretic separation to the following 30 information from Example 2.

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Claims (8)

1. Blood coagulation-promoting preparation based on human proteins containing the coagulation factors 2. Preparation according to claim 1, characterized in that the protein with factor IX activity at a NaCl concentration of 0.1 to 0.5 (molar) and the 35 'protein with FEIB activity at a NaCl concentration of 0 , 3 to 0.5 (molar) eluted; the maximum factor IX activity eluting at 0.3 (molar) and the maximum v -22- * FEIB activity at 0.4 (molar). 3. Preparation. According to claims 1 and 2, characterized in that the FEIB activity after one hour 4. Preparation according to claims 1 and 2, characterized * indicates that the factor IX activity after at least 10 hours of incubation in factor IX deficient plasma remains at least 50%. 5. Preparation according to claims 1 to 4, characterized in that it contains inter-alpha 15 has trypsin inhibitor (ITI) of 0.05 to 5 mg per FEIBA unit. 5 Incubation in factor VIII inhibitor plasma remains at least 50%. e 5 II, VII, IX and X and a factor VIII inhibitor bypass activity, characterized in that it is free from thrombogenic activity up to at least 2 units of FEIBA per kg of rabbit in the thrombosis-inducing activity test Wessler, 10. that it is free from kallikrein activity and free from precallikrein activator activity - measured in an aqueous solution of the preparation with a FEIBA concentration of up to at least 10 units per ml, 15. that it is affinity chromatographically on dextran sulfate - Agarose can be separated using a NaCl gradient in such a way that the protein with factor iX activity elutes at a lower NaCl concentration than the protein with FEIB activity, 20. that the protein with factor IX activity and the protein with FEIB activity containing eluates in electrophoretic separation contain a and ^ globulins, the separation curve in the a region being a main peak corresponding to 60 to 80% of the total protein, e an adjoining shoulder of 10 to 20% of the total protein and a weakly pronounced peak in the ^ -globulin region following the shoulder-shaped course of the separation curve, corresponding to a content of 10 to 20% * 30. of the total protein. 6. A method for producing a blood coagulation-promoting preparation according to claims 1 to 5, characterized in <20 that human plasma is treated with sulfated, highly polymeric carbohydrates and / or with basic ion exchangers and the protein mixture is adsorbed with generated FEIB activity, whereupon it is' eluted and concentration is gained. 25th '7. A method according to claim 6, characterized in that the plasma is first briefly treated with sulfated high polymer carbohydrates, the protein mixture with generated FEIB activity is then adsorbed on an ion exchanger based on dextran and immediately * eluted and concentrated thereon. 8. The method according to claim 6, characterized in that the plasma is treated with an ion exchanger based on dextran 35 and, after at least two hours of exposure, the protein mixture adsorbed on the ion exchanger is eluted with generated FEIB activity and then concentrated.